Phospholipase A2 (PLA2) plays a central role in membrane phospholipid homeostasis, phospholipid remodeling, and general phospholipid metabolism. It also catalyzes the key first step in the biosynthesis of the eicosanoids, including the prostaglandins and leukotrienes, and is implicated in signal transduction. The long term objectives of this proposal are (i) to understand the detailed mechanism of action of cobra venom (N. naja naja) PLA2 and to use it as a model for lipolytic enzymes in general and (ii) to identify, purify, and characterize the phospholipase responsible for the release of arachidonic acid in the biosynthesis of eicosanoids. Specific studies on the cobra venom PLA2 are aimed at evaluating the tertiary structure of the enzyme; defining the precise role of the "activator" site in the mechanism; determining whether the functionally active enzyme subunit is a monomer or dimer; determining the amino acid residues important for catalysis and activation; determining how lipid induces aggregation of the enzyme; and determining the nature of phospholipid binding to the enzyme. These aims will be accomplished by a combination of biochemical techniques, including kinetic analysis, inhibition studies, chemical modification, and by the molecular biological techniques of cloning and site-directed mutagenesis. The study of eicosanoid biosynthesis will focus on the phospholipases found in the macrophage-like P388D1 cell line. The specific aims include continuing to investigate the various lipolytic enzymes in these cells and their connection to eicosanoid biosynthesis; to identify and express the gene for the Ca2+-dependent membrane-associated PLA2, the prime candidate for the first enzyme in the eicosanoid cascade; to clone and produce this enzyme in sufficient quantities to use in the same type of kinetic and biochemical studies employed in the work with the cobra venom enzyme; and to determine how this enzyme is regulated.